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1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany
2 Genomik Netzwerk Göttingen, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany
3 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany
4 Institut für Biologie, Humboldt-Universität Berlin, Chausseestrasse 117, 10115 Berlin, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.
The GenBank accession numbers for the nucleotide sequences reported in this paper are AY489113 (Ralstonia eutropha H16 phaP3), AY489114 (phaP4) and AY489115 (phaZ5).
The following are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org: an alignment of the amino-acid sequences of phasin homologues of R. eutropha H16 PhaP1 in supplementary Fig. I; the regions adjacent to phaP1, phaP2, phaP3 and phaP4 in the R. eutropha H16 genome in supplementary Fig. II; the alignment of multiple Re1052 digestion fragments with the proposed H16 sequence in supplementary Fig. III; the molecular masses of R. eutropha phaP1 knock-out mutant Re1052 granule proteome fragments by MALDI-TOF in supplementary Table I; the similarities of the five phaZ homologues of R. eutropha in supplementary Table II; the similarities of the four phaP homologues of R. eutropha in supplementary Table III.
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